Apoptosis (2010) 15:1507–1516 DOI 10.1007/s10495-010-0532-6 ORIGINAL PAPER Multiwalled carbon nanotubes activate NF-jB and AP-1 signaling pathways to induce apoptosis in rat lung epithelial cells Prabakaran Ravichandran • Sudhakar Baluchamy • Bindhu Sadanandan • Ramya Gopikrishnan • Santosh Biradar Vani Ramesh • Joseph C. Hall • Govindarajan T. Ramesh • Published online: 8 August 2010 Ó Springer Science+Business Media, LLC 2010 Abstract Our previous report on multiwall carbon nanotubes (MWCNT) has demonstrated the generation of reactive radicals and depletion of intracellular antioxidants which in turn cause cell death through activation of caspases. The molecular mechanism of cellular death due to MWCNT is not clear yet. In this study, we investigated the signaling pathways implicated in MWCNT-induced apoptosis in rat lung epithelial cells. First, we assessed the DNA damage in response to MWCNT treatment and showed the significant DNA damage as compared to control. The collapse of the mitochondrial membrane integrity, release of cytochrome c into the cytosol, reduction in cellular ATP content, increased levels of mitochondrial apoptogenic factor and activation and nuclear translocation of NF-jB were observed in MWCNT treated cells. In addition, a time-dependent induction of phosphorylated IjBa and its degradation were detected in cells exposed to MWCNT. Furthermore, MWCNT activated several death related proteins including apoptosis inducing factor, p53, p21 and bax. Together, our results suggest that signaling pathways such as NF-jB and AP-1 are activated upon MWCNT treatment for cellular cytotoxicity. P. Ravichandran
S. Baluchamy
B. Sadanandan
R. Gopikrishnan
S. Biradar
V. Ramesh
J. C. Hall
G. T. Ramesh (&) Molecular Toxicology Laboratory, Center for Biotechnology and Biomedical Sciences, Department of Biology, Norfolk State University, Norfolk, VA 23504, USA e-mail: gtramesh@nsu.edu P. Ravichandran
S. Baluchamy
B. Sadanandan
R. Gopikrishnan
S. Biradar
V. Ramesh
J. C. Hall
G. T. Ramesh Department of Biology, Texas Southern University, Houston, TX 77004, USA Keywords Apoptosis
Cytochrome c
NF-jB
AP-1
p53
DNA damage Introduction Carbon Nano Tubes (CNTs) are emerging as an important new class of multifunctional building blocks in the field of nanotechnology [1]. The demand for large scale production of multiwall carbon nanotubes (MWCNT) is increasing, because of their potential applications [2–4]. CNTs of inhalable size are aerosolized during mechanical agitation [5]. Besides the industrial production, CNTs are generated by burning methane, propane, and natural gas [6]. So, the professional and public exposure to MWCNTs is expected to increase significantly in the coming years. Various toxicological animal studies using CNTs demonstrate that pulmonary deposition of singlewall carbon nanotubes (SWCNT) or MWCNT which causes acute pulmonary inflammation as well as chronic responses such as fibrosis [7–10]. Pulmonary deposition of SWCNT or MWCNT results in a rapid release of inflammatory mediators, activated blood cells, and thrombogenic proteins into the systemic circulation which may induce endothelial dysfunction [11]. An in vitro study of MWCNT indicates that they activate genes involved in stress responses, cellular transport, metabolism, and cell cycle regulation in human skin fibroblasts [12], cause injure to plasma membrane of macrophages [13] and alter the paracellular permeability of human airway epithelial cells [14]. Taken together, the evidence from carbon nanotube toxicity studies indicate the necessity to systematically define the basic mechanism(s) underlying their toxicity. Recently, we have reported that MWCNTs induced oxidative stress and apoptosis through caspase activation in 123 1508 rat lung epithelial cell (LE) [15]. Alterations in intracellular redox reactions have been shown to activate signaling cascades, which regulate early response genes that are involved in protective or reparative mechanisms. Transcription factors, such as nuclear factor-kappa B (NF-jB) and activator protein-1 (AP-1), are considered stress response transcription factors, which regulate the expression of a variety of downstream target genes [16]. The AP-1 heterodimers are constitutively localized within the nucleus and transactivation of AP-1 is achieved through phosphorylation of its activation domain by c-Jun N-terminal kinase (JNK) [17]. In contrast, NF-jB remains inactive in the cytoplasm, through interaction with specific inhibitors, IjBs [18]. NF-jB is activated in response to proinflammatory stimuli; the IjBs are rapidly phosphorylated and resulting in the release of free NF-jB dimers, which translocate to the nucleus to induce transcription of target genes [19]. In addition, it has been reported that the AP-1 and NF-rB are regulated by MAPK under oxidative stress [20, 21]. p53 regulates multiple responses to genotoxic stress by transcriptional activation or repression of a number of genes including genes involved in cell cycle control (p21WAF1/Cip1), DNA repair (gadd45), and apoptosis (e.g., Bax, Bcl2 and survivin) [22]. The present study was aimed to further understand the molecular mechanism particularly, signaling pathways responsible for MWCNT mediated cytotoxicity in rat LE cells. Here, we show an involvement of mitochondria, nuclear transcription factors and cell death regulatory proteins during MWCNT treatment. Experimental procedures Materials Penicillin, Streptomycin, Dulbecco’s modified Eagle’s medium, and Fetal Bovine Serum were purchased from Atlanta Biologicals, Inc (Atlanta, GA). The Live/Dead assay kit was purchased from Molecular Probes (Eugene, OR). The mitochondria isolation kit and M-PER Mammalian Protein Extraction Reagent were purchased from Thermo Fisher Scientific (Rockford, IL). Antibodies against c-jun, c-myc, p21, p53, b-actin, cytochrome c oxidase assay kit (CYTOCOX1) and MWCNT (OD 9 length 6–13 nm 9 2.5–20 lm) were purchased from Sigma-Aldrich chemicals (MI, USA). TransAM NF-jB family assay kit, Trans AM AP-1 ELISA kit and nuclear extraction kit were purchased from Active motive, (Carlsbad, CA). ApoSensor ADP/ATP ratio assay kit was obtained from Bio-Vision (Mountain View, CA). Antibodies against cytochrome c, p65, p50, IrBa, c-fos, AIF, Bax, CRM1 and Tubulin were purchased from Santa Cruz Biotechnology, (Santa Cruz, CA). Antibody against anti-P42/44 phosphorylated MAPK antibody 123 Apoptosis (2010) 15:1507–1516 was purchased from Cell Signaling Technologies (Beverly, MA). Anti-rabbit and anti mouse antibodies were purchased from Biorad, (Hercules, CA). Cell lines and treatment Rat LE cells (RL 65, CRL-10354) were purchased from American Type Culture Collection (Manassas, VA) and were cultured in DMEM with 10% FBS, 100 IU/ml of penicillin, and 100 lg/ml of streptomycin and incubated at 37°C in a humidified chamber with 5% CO2. For all studies, MWCNT stock was prepared by dissolving in DMF and therefore in all the control experiments, cells were treated with equivalent volume of DMF. Cytotoxicity assay (live and dead cell assay) The cytotoxic effect of MWCNT was determined by the Live/Dead assay kit [19]. The kit provides a two-color fluorescence cell viability assay that is based on the simultaneous determination of live and dead cells with two probes that measure recognized parameters of cell viability. It is generally faster, less expensive, safer and a more sensitive indicator of cytotoxic events than other methods. Live cells are distinguished by the presence of ubiquitous intracellular esterase activity, determined by the enzymatic conversion of the virtually non fluorescent cell-permeant calcein AM to the intensely fluorescent calcein. The polyanionic dye calcein is well retained within live cells, producing an intense uniform green fluorescence in live cells (ex/em * 495 nm/*515 nm). Ethidium-1 (EthD-1) enters cells with damaged membranes and undergoes several fold enhancement of fluorescence upon binding to nucleic acids, thereby producing a bright red fluorescence in dead cells (ex/em * 495 nm/*635 nm). EthD-1 is excluded by the intact plasma membrane of live cells. The determination of cell viability depends on physical and biochemical properties of cells. Background fluorescence levels are inherently low with this assay technique because the dyes are virtually non fluorescent before interacting with cells. Briefly, 1 9 105 cells were treated with 5 lg of MWCNT and cultured. Following that cells were stained at 12 and 24 h of treatment with Live/Dead reagent (5 lM ethidium homodimer, 5 lM calcein-AM) and incubated at 37°C for 30 min. Finally, cells were analyzed using Nikon Fluorescence microscope (Nis Element, Nikon Instruments Inc, Melville). Apoptotic DNA ladder analysis Apoptotic DNA ladder analysis was performed as described earlier [23]. LE cells were stimulated with different Apoptosis (2010) 15:1507–1516 concentration of MWCNT and cultured for 24 h. Following that, the cells were harvested and genomic DNA was isolated using Quick Apoptotic DNA ladder kit (Invitrogen) according to the procedure specified by manufacturer. Briefly, equal number of cells from 24 h of MWCNT treated (different concentration) and control were homogenized in TE buffer followed by mixing with Enzyme A solution and incubated at 37°C for 10 min. Enzyme B solution was added to the enzyme A mix and incubated for additional 30 min at 50°C. To this, one tenth volume of ammonium acetate and 2.5 fold cold ethanol were added and precipitated at -20°C for 15 min followed by centrifugation to get the DNA pellet. The pellet was washed with 70% cold ethanol and centrifuged again. Finally, the DNA was air-dried and resuspended in DNA suspension buffer and analyzed on 1.2% agarose gel. Measurement of ATP and ADP/ATP ratio The changes in ADP-to-ATP ratios have been used to conveniently differentiate apoptotic from necrotic cell death [24]. The assay was performed to detect the ATP levels and ADP-to-ATP ratio [25]. Briefly, the assay utilizes the enzyme luciferase to catalyze the formation of light from ATP and luciferin. ADP levels are measured by conversion to ATP that is subsequently detected using the same reaction. Luminescence was measured with a Luminoskan Ascent luminometer (Thermo Electron Inc., Milford, MA). Isolation of mitochondria Mitochondrial isolation was carried out according to the mitochondria isolation kit (PIERCE, Rockford, IL). Briefly, 1 9 106 LE cells were treated with different concentration of MWCNT and cultured for 24 h. The cells were harvested after the treatment and centrifuged at 850 g for 2 min. The pellet was first resuspended in 800 ll of reagent A, incubated for 2 min on ice followed by addition of reagent B (10 ll) and incubation for another 5 min on ice. Next, reagent C (800 ll) was added to reagent A and B mix and inverted for several times to mix. Finally, the solution was centrifuged at 700 g for 10 min at 4°C, and the pellet was used for crude nuclei fraction. The supernatant was centrifuged at 12,0009g for 15 min at 4°C and transferred to a new tube for the post-mitochondrial supernatant fraction. The pellet was washed with 500 ll of regent C, and used as mitochondrial fraction. Cytochrome c assay The Cytochrome c Oxidase (COX) activity was measured as described earlier [26]. The colorimetric assay is based 1509 on the decrease in absorbance at 550 nm of ferrocytochrome c caused by its oxidation to ferricytochrome c by COX. Mitochondria (approximately 0.4 mg protein) were sonicated, and added to a standard 1 ml cuvette with assay buffer (10 mM Tris–HCl, pH 7.0, containing 120 mM KCl) to a final volume of 950 ll. To begin the reaction, 50 ll of ferrocytochrome c (0.22 mM) substrate solution was added. The change in absorbance at 550 nm was read immediately. The activity of COX was calculated using an extinction coefficient of 21.84. Measurement of the outer membrane integrity The mitochondrial outer membrane integrity was analyzed according to manufacturers’ protocol [Sigma, CYTOCOX1]. Briefly, two parallel samples of the mitochondrial suspension containing 0.2 mg protein/ml were diluted with and without n-dodecyl b-D-maltoside using enzyme dilution (10 mM Tris–HCl, pH 7.0 containing 250 mM sucrose) buffer. After incubation for 10 min at 4°C, 2 lg of mitochondrial protein was used for assaying COX activity. The change in absorbance at 550 nm was read immediately. TransAM NF-jB activation assay TransAM assays of all five NF-jB family members were measured as described previously [27]. Briefly, 1 9 106 LE cells were stimulated with various concentration of MWCNT for 24 h at 37°C. Ten microgram of nuclear protein was incubated for 1 h in a 96-well plate coated with an oligonucleotide that contains the NF-jB consensus site (50 -GGGACTTTCC-30 ) to which activated NF-jB factors in nuclear extracts specifically bind. The primary antibodies used in the TransAM NF-jB Kit recognize an accessible epitope on the NF-jB proteins upon DNA binding. After incubation for 1 h with a secondary HRP-conjugated antibody, specific binding was detected by colorimetric estimation on a spectrophotometer at 450 nm with a reference wavelength of 655 nm. AP-1 activation assay The effect of MWCNT on AP-1 family proteins were investigated by stimulating LE cells with 5 lg/ml of MWCNT particles for 24 h. 10 lg of nuclear extract obtained from control and treated cells were analyzed by Trans AP-1 assay as described [28]. Nuclear proteins were incubated with consensus TRE-oligonucleotides (50 -TGAGTCA-30 ) immobilized on 96-well assay plates and probed with antibodies specific to members of the Fos (c-Fos, FosB, Fra-1, and Fra-2) and Jun related protein (JunB, JunD, and c-Jun). After incubation for 1 h with a 123 1510 secondary HRP-conjugated antibody, specific binding was detected by colorimetric estimation on a spectrophotometer at 450 nm with a reference wavelength of 655 nm. Apoptosis (2010) 15:1507–1516 a MWCNT Treated untreated 12 h 24 h Western blot analysis 0% b 29% 71% Cont 0.1µg 2.5µg MWCNT 5µg Cytoplasmic, nuclear and whole cell extracts were prepared from LE cell treated with 5 lg/ml of MWCNT at different time points as indicated. Samples containing 50 lg of cellular protein were subjected to standard western blot analysis [19, 29]. Briefly, equivalent amounts of proteins were separated using 10% SDS-PAGE and electro transferred to polyvinylidene diflouride membrane. Immunoblotting was performed by blocking overnight with 5% nonfat milk in PBS-0.1% Tween, probed with appropriate primary antibody followed by secondary antibody conjugated with horse radish peroxidase and developed using chemiluminescence reagent (GE Healthcare, Buckinghamshire, UK). M bp 12216 5090 3054 2036 1636 1018 506 Statistical analysis For significant changes, data were analyzed by student’s t test. A P value less than 0.05 was considered statistically significant. Results MWCNT induce apoptosis in rat LE cells In order to check the toxicity effect of MWCNT on rat LE cells, cell viability assay was performed. Figure 1a shows a time dependent inhibition of cell viability in 5 lg of MWCNT treated LE cells as compared to control cells. The percentage of dead cells was progressively increased from 29 to 71% across a time span of 12–24 h. In our previous report, we have showed that MWCNT treated cells show a strong intensity of fluorescence in TUNEL assay, indicating a massive DNA breakage as compared to control cells [15]. To further confirm our MWCNT induced apoptosis, the DNA fragmentation assay was performed. Figure 1b shows much higher levels of DNA fragmentation in 5 lg MWCNT treated cells as compared to control and other lower doses (Fig. 1b). Therefore, we used 5 lg of MWCNT in most of our experiments. Measurement of ATP and ADP/ATP ratio Since a cellular redox change may decrease energy production in the form of ATP from mitochondria, we examined intracellular ATP levels. Figure 2a shows a marked decline in cellular ATP content. The ATP level alone appears to be insufficient to determine whether LE 123 Fig. 1 Effect of MWCNT on cell viability and cell death in LE cells. a Time-dependent inhibition of cell viability by MWCNT. Approximately 105 LE cells were treated with 5 lg MWCNT and cultured. The cell viability was assayed using ‘‘Live/Dead cell assay kit’’ and the number of live and dead cells were counted at 12 and 24 h and photographed. The percentage of dead cells is indicated below. b Dose dependent DNA fragmentation by MWCNT. Equal amount of LE cells were incubated with 0.1, 2.5 and 5 lg of MWCNT and cultured for 24 h. Genomic DNA was isolated using apoptotic DNA ladder kit and analyzed on 1.2% agarose gel cells undergo apoptosis or necrosis in response to MWCNT particles. Therefore, we further investigated the ratio of cellular ADP to ATP. Our results show that ADP-to-ATP ratio was significantly increased in MWCNT treated cells as compared to control suggesting that MWCNT treated cells undergo apoptosis rather than necrosis (Fig. 2b). MWCNT induce cytochrome c release Dysfunction of mitochondrial membrane increases the permeability of the mitochondrial membrane and subsequently promotes the release of cytochrome c oxidase (COX) enzyme. To determine whether the change in mitochondrial membrane lead to release of COX, the cytosolic and mitochondrial fractions were analyzed from MWCNT treated cells. No significant level of COX activity was detected in the cytosolic extracts from untreated control cells whereas the MWCNT triggered COX release to Apoptosis (2010) 15:1507–1516 1511 b 30 0.3 25 0.24 20 15 * * 10 * 5 ADP/ATP ratio ATP production ( µg / mg protein ) a 0.18 0.12 0.06 0 0 0 0.5 1 3 12 24 36 Time (Hours) 0 0.5 1 3 12 24 36 Time (Hours) Fig. 2 Effect of MWCNT on ATP level and ADP/ATP ratio. a MWCNT decreases ATP level in LE cells. LE cells were stimulated with 5 lg of MWCNT and lysate was prepared at various time intervals as indicated in the figure and ATP levels were determined as described in ‘‘Experimental procedures’’ section. *P \ 0.05 significance as estimated by student’s t test. b Time-dependent induction of apoptosis analyzed by ADP/ATP ratio in 5 lg MWCNT treated cells the cytosol (Fig. 3a, left panel). The amount of COX activity increase in cytosol is time dependent. This is also confirmed by western blot showing more cytochrome c proteins in MWCNT treated cells (Fig. 3c). Conversely, we also found that there was a significant decrease in the mitochondrial COX activity suggesting the leakage of COX from mitochondria into cytosol (Fig. 3a, right panel). To reconfirm the leakage of cytochrome-c oxidase, the integrity of the mitochondrial outer membrane is assessed in the presence and absence of the detergent, n-dodecyl b-D-maltoside. The result revealed a dose dependent decrease in the integrity of outer membrane at concentration as low as 2.5 lg/ml (Fig. 3b). Next, we determined whether mitochondrial apoptogenic factor, apoptosis inducing factor (AIF) is also involved in MWCNT induced apoptosis, we performed western blot analysis on the nuclear extract and show an increased level of AIF protein after 3 h of MWCNT treatment, and the level was further increased in the later time points (Fig. 3d). explore, whether other subunits of NF-jB such as p52, c-Rel, and Rel B also delocalize to the nucleus upon MWCNT treatment, we performed TransAM assay using same nuclear extract used for p50/p65. As shown in Fig. 4d, p65 and p50 and c-rel were activated at much higher level as compared to other subunits; p52 and rel-B in 5 lg MWCNT treated cells. MWCNT activates NF-jB MWCNT induce mitogen-activated protein kinase (MAPK) In order to study the redox responsiveness of the transcriptional regulator NF-jB in LE cells, we used TransAM assay to assess activation by nuclear binding activity. The result clearly showed an activation of p65 at concentrations as low as 2.5 lg of MWCNT and the increase was dosedependent (Fig. 4a). The localization of p50 and p65, which are known to translocate to the nucleus upon activation was assessed quantitatively in nuclear fractions of MWCNT treated cells. Quantification of NF-jB activation in MWCNT exposed cells showed a time-dependent induction of p65 and p50 with maximum activity at 24 h (Fig. 4b). In addition, our western blot shows an increased level of p50/65 proteins in the nuclear extracts of MWCNT treated cells as compared to control (Fig. 4c). Next, to IjBa degradation in response to MWCNT The translocation of NF-jB to the nucleus is preceded by phosphorylation and proteolytic degradation of IjBa in the cytosol. Therefore, we determined IjBa degradation in MWCNT treated cells. Cytoplasmic extract from 5 lg MWCNT treated cells show the significant reduction of IjBa protein at 2 h of post treatment and reach the maximum reduction thereafter (Fig. 5). Our results show the perfect correlation between the activation of NF-jB and the degradation of IjBa. Published reports show that mitogen-activated protein kinases (MAPK) can participate in the regulation of NF-jB transcriptional activity [30]. Therefore, we performed an immunoblot analysis for phosphorylated and total MAPK proteins in MWCNT treated cells and showed a dose dependent increased level of phosphorylated p42 and p44 MAPK as compared to control. As expected, the total MAPK levels were not altered in the same extract (Fig. 6). MWCNT induce AP-1 Activator Protein-1 (AP-1) is composed of hetero- or homodimer subunits of proteins fos, jun, jun dimerization 123 1512 Apoptosis (2010) 15:1507–1516 Cytosol * 0.002 0.0015 * 0.001 0.0005 0 Control 0.5 1 Mitochondria 0.06 0.0025 COX activity (unit/min/ml) COX activity (unit/min/ml) a 3 6 0.05 0.04 0.03 * 0.02 * 0.01 0 12 Control 0.5 1 Time (Hours) 1.2 Cytoplasmic c 1 0.8 0.6 0.4 * 0 Control 0.1 5 2.5 6 12 10 MWCNT Treated Control 12 24 36 (Time in h) CytochromeC Tubulin MWCNT Treated d * 0.2 Nuclear % of undamaged outer membrane (folds) b 3 Time (Hours) Control 1 3 6 12 36 (Time in h) 24 AIF CRM1 MWCNT (µg/ml) Fig. 3 Effect of MWCNT on cytochrome-c oxidase (COX). a MWCNT reduces cytochrome c level in mitochondria. LE cells were treated with 5 lg of MWCNT and the cytoplasm and mitochondrial fractions were extracted at different time intervals as indicated and COX activity was detected. The COX activity expressed as unit/min/ml from mitochondrial and cytosolic fractions are shown right and left panel, respectively. b MWCNT cause mitochondrial outer membrane damage. LE cells were treated with different concentration of MWCNT and the percentage of 123 4.5 * 4 3.5 * 3 2.5 2 1.5 1 0.5 0 Control 0.1 2.5 5 NFκB activation ( OD 450 nm ) b a 0.04 Control 0.035 Nuclear 0.02 0.015 0.01 0.005 0 0.5 1 2 12 24 (Time in h) p65 p50 CRM1 NFκB activation (OD 450 nm ) Control 6 3 Time in h MWCNT Treated p50 0.025 10 d c P65 0.03 MWCNT (µg/ml) Nuclear p 65 activation ( OD 450 nm ) Fig. 4 Effect of MWCNT on NF-jB activation. a LE cells were stimulated with different concentration of MWCNT for 12 h and nuclear extracts were prepared and used for p65 quantification as described in ‘‘Experimental procedures’’ section. b LE cells were treated with 5 lg of MWCNT and translocation of p50/65 into the nucleus was detected at various time intervals (see ‘‘Experimental procedures’’ section for details). c Immunoblot of p50 and p65 proteins in 5 lg MWCNT treated nuclear extracts; CRM1 was used as a loading control. d LE cells were treated with 5 lg of MWCNT for 12 h and DNA binding activities for NF-jB family members are shown *P \ 0.05 significance as estimated by student’s t test mitochondria damage in outer membrane was assayed after 12 h of post treatment (see ‘‘Experimental procedures’’ section for details). c Western blot showing cytochrome c protein in 5 lg MWCNT treated cytoplasmic fraction. d AIF protein level at different time points of 5 lg MWCNT treated nuclear fraction. Tubulin and CRM1 were used as loading controls for cytoplasmic and nuclear extracts respectively. The results (a, b) are shown as a representative of three independent experiments. *P \ 0.05 significance as estimated by student’s t test 1.4 1.2 * * 1 0.8 0.6 0.4 0.2 0 * 6 12 24 Apoptosis (2010) 15:1507–1516 1513 MWCNT Treated (Time in h) 0.5 1 2 6 12 I Bα Tubulin 3.5 Relative protein levels 1.2 24 3 2.5 2 AP-1 activation ( OD 450 / 650 ) Cytoplasmic 0 a Untreated 0.9 ∗ Treated ∗ ∗ 0.6 ∗ ∗ 0.3 1.5 0 1 c-fos fos B fra-1 fra-2 c-jun jun D Jun B 0.5 0 0 0.5 1 2 6 12 b 24 MWCNT Treated Control 6 12 (Time in h) 24 Time in h Fig. 5 Effect of MWCNT on IrBa degradation. LE cells were treated with 5 lg of MWCNT and cytosol fractions were prepared at different time intervals as indicated and IrBa proteins were detected by immune blotting. The same blots were reprobed with anti-tubulin for equal loading. The relative protein levels were quantified using densitometric analysis MWCNT (µg/ml) 0 0.5 5 10 44 Phosphorylated MAPK 42 Nuclear c-fos c-Jun CRM1 Fig. 7 MWCNT activates AP-1 family. a LE cells were treated with 5 lg of MWCNT for 12 h and nuclear extracts were prepared and DNA binding activities for AP-1 family members were detected as described in ‘‘Experimental procedures’’ section. b Nuclear extracts were prepared from different time points of 5 lg MWCNT treated LE cells and c-fos and c-jun protein levels were detected using specific antibodies. The blots were stripped off and reprobed with anti CRM1 antibody for loading control MWCNT Treated Total MAPK -Actin Control 12 24 36 (Time in h) p53 Fig. 6 Effect of MWCNT on MAPK activation. LE cells were treated with increasing concentrations of MWNCT for 24 h and lysates were prepared. 50 lg of proteins were resolved on SDS-PAGE and phosphorylated MAPK and total MAPK were detected using specific antibodies. The blots were stripped off and reprobed with b-actin to ensure equal loading partner (JDP), and activating transcription factor (ATF) families [28]. Trans Ap-1 family assay shows much higher levels of c-fos and c-jun as compared to other subunits in 5 lg MWCNT treated nuclear extracts (Fig. 7a). To further confirm the activation of c-fos and c-jun subunits, western blot analysis was performed from nuclear extract isolated at different time points of 5 lg MWCNT treated cells and showed the time dependent increased levels of c-fos and c-jun proteins (Fig. 7b). Effect of MWCNT on tumor suppressor proteins Previously, it has been reported that the tumor suppressor protein p53 plays a role in the control of cell growth and p21 Bax β-Actin Fig. 8 Effect of MWNCT on p53, p21 and bax. LE cells were treated with 5 lg of MWNCT for various time points as indicated and lysates were prepared. 50 lg of proteins were resolved on SDS-PAGE and p53, p21, and bax proteins were detected using specific antibodies. The blots were stripped off and reprobed with b-actin antibody for loading control apoptosis. The p53 is also partly regulated by NF-jB [31]. In order to check whether p53 protein is altered in MWCNT treated cells, we performed immune-blot for p53 and its target p21. Figure 8 shows an increased level of p53; p21 and bax proteins at 12 h of 5 lg MWCNT treated cells as compared to control cells. Notably, the increase was not altered much at later time points (24 and 36 h). 123 1514 Discussion MWCNTs are seen as having a huge potential outcome in many areas of research and application [32]. With their morphology similar to asbestos fibers, the assessment of the respiratory toxicity drew attention of many scientists. Therefore, the toxicological studies on MWCNT require detailed evaluation in order to set up minimal standards to avoid health calamities in near future. In this report, we show the dose dependent inhibition of cell viability in MWCNT treated rat LE cells. Previously, our group has shown that SWCNT treatment induced oxidative stress and activated NF-jB in human keratinocytes [19]. Similar findings were also reported with SWCNT on the proliferation of HEK293 kidney epithelial cells [33], MWCNTs on skin epithelial and A549 cells [34, 35]. Free radicals can chemically alter DNA bases and cause DNA damage within the cell. DNA fragmentation and condensation is a hallmark of apoptosis [36]. Here, we observed MWCNT exposed cells displayed nuclear fragmentations which correlate with previous findings [37]. It has been shown that a moderate increase in the ADP-to-ATP ratio indicates apoptosis, whereas a higher ADP-to-ATP ratio points toward necrosis [38]. Also, mitochondrial transmembrane potential disruption, release of mitochondrial death mediators and subsequent activation of caspases are shown to be involved in ROS-mediated apoptosis pathway [39]. Permeabilization of the outer mitochondrial membrane (OMM) leads to the cytosolic release of multiple proapoptotic intermembrane proteins, including COX, initiating cell death. Here, we reported that MWCNT cause disruption of mitochondrial membrane integrity, release of COX from mitochondria to cytosol (Fig. 3). Our present findings support the earlier studies which demonstrated that the increase in ROS production results in release of COX from the mitochondria in LE and pancreatic islet b-cell apoptosis [39, 40]. Our result also demonstrated that MWCNT induced translocation and up regulation of AIF in LE cells which support earlier observation [41]. Results from this study clearly show that MWCNT activates both transcriptional factors AP-1 and NF-jB in LE cells for programmed cell death. It has been well documented that both AP-1 and NF-jB are considered as stress responsive transcription factors that govern the expression of proinflammatory and cytotoxic genes [42]. Our group and others have reported similar activation of NF-jB upon oxidative stress in HaCaT and BEAS-2B cells [19, 43]. The MWCNT-induced NF-jB activation was accompanied by characteristic IjBa degradation in a time-dependent manner. Previous reports from our laboratory showed that metals such as lead, manganese (Mn2?) and arsenite induced NF-jB activation and corresponding degradation of IjBa in PC12 and 123 Apoptosis (2010) 15:1507–1516 mesencephalic cell lines, respectively, [44, 45]. NF-jB heterodimers and homodimers are present in the cytoplasm of most cells as inactive complexes with inhibitory IjB proteins. Various exogenous stimuli including oxidant can trigger reactions that lead to degradation of the bound IjB protein and a rapid translocation of NF-jB to the nucleus, where it binds to NF-jB consensus sequences and activates the transcription of several genes [46]. The phosphorylation of IjBa is accomplished by a specific IjB kinase (IKK) and this is further activated by several upstream kinases such as MAPK kinases [19]. Here, our observation shows that MWCNT induced dose dependent phosphorylation of MAPK kinase which could in turn transactivate NF-jB. Similar NF-jB activation has been described earlier through MAPK for SWCNT in HaCaT cells [19] and interleukin 8 in U937 cells [47]. Finally, MWCNT exposure resulted in p53 accumulation and its two well-studied transcriptional targets, p21 and bax. At present, we do not know the exact role of p21 and p53 in MWCNT–mediated cell death. Experiments on in vivo mouse model system are underway to investigate whether the same signaling pathways are activated during MWCNT treatment or not. In summary, our results add to the growing evidence showing that MWCNT induced oxidative stress which in turn trigger nuclear transcription factor AP-1 and NF-jB and release cytochrome c from mitochondria for cell death. Moreover, this is the first report to show the molecular events associated with MWCNT mediated cellular cytotoxicity. Acknowledgments This work was supported by NASA funding NNX08BA47A: NCC-1-02038: NIH 1P20MD001822-1. References 1. DaiLy (2006) Carbon nanotechnology. Elsevier, Amsterdam 2. Baughman RH, Zakhidov AA, de Heer WA (2002) Carbon nanotubes-the route toward applications. Science 297:787–792 3. 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